Soil Biology & Biochemistry 81 (2015) 275e281

Contents lists available at ScienceDirect

Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Mechanisms of soil acidification reducing bacterial diversity

Ximei Zhang a, b, Wei Liu c, d, Guangming Zhang e, Lin Jiang b, Xingguo Han a, e, *
a
State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China
b
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA
c
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
d
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
e
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

a r t i c l e i n f o a b s t r a c t

Article history: A central goal in soil microbial ecology research is to identify the biodiversity patterns and reveal the
Received 21 May 2014 underlying mechanisms. Long-term soil acidification is known to reduce soil bacterial diversity, but the
Received in revised form mechanisms responsible for this pattern have not been well explored. Soil acidification may reduce
30 October 2014
bacterial richness through ecological filtering (EF). In contrast, two types of processes may promote the
Accepted 5 November 2014
Available online 18 November 2014
maintenance of bacterial richness: species may adapt to the acidic pressure through evolution, and
endemic species already adapted to the acidic pressure can colonize the acidified soils through dispersal.
To identify the relative contribution of EF and evolution/dispersal (ED), we collected soils with a pH range
Keywords:
Microbial diversity
of 4e7 from different ecosystems, conducted an acidification experiment with a similar pH range in a
Soil acidification neutral soil, and proposed a conceptual framework that could distinguish the three potential types of
Ecological filtering mechanism (neither EF nor ED operate; EF operates alone; ED counteracts some effect of EF). We found
Environmental filtering that the entire bacterial domain was driven by the third type of mechanism, with ED counteracting about
Evolutionary adaptation 42.4% (95% confidence interval: 32.7e50.4%) effect of EF. Meanwhile, different bacterial phyla/classes
Dispersal were governed by different types of mechanisms, and the dominant was the third type. Our results
highlight the importance of both ecological and evolutionary mechanisms for regulating soil bacterial
communities under environmental changes.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Soil microbial ecology research is still at the descriptive stage
(Horner-Devine et al., 2004; Martiny et al., 2006). Most previous
studies usually aimed to reveal various types of biodiversity pat-
terns, but the underlying mechanisms were often not fully explored
(Prosser et al., 2007; Hanson et al., 2012). While precipitation and
temperature are the primary drivers of plant diversity across
different terrestrial ecosystems, soil pH has been found to be a key
factor shaping bacterial diversity (Fierer and Jackson, 2006; Lauber
et al., 2009; Rousk et al., 2010). For example, Fierer and Jackson
(2006) found that soil pH alone accounts for more than 70% vari-
ation in bacterial diversity across different terrestrial ecosystems,
and other ecological factors, such as precipitation and soil nitrogen
* Corresponding author. State Key Laboratory of Forest and Soil Ecology, Institute
of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. Tel.: þ86
139 1131 7831.
E-mail address: xghan@ibcas.ac.cn (X. Han).
content, account for only a small part of variation. They also found
that as soil pH increases (from acidic to alkaline), bacterial diversity
first increases then declines. The relationship between soil pH and
bacterial diversity is arguably one of the most important patterns in
microbial ecology (Fierer and Jackson, 2006; Jones et al., 2009).
It is generally agreed that all terrestrial ecosystems and their
biological components are originated from the ocean (Nisbet and
Sleep, 2001; Martin et al., 2008). Because the pH of the primitive
ocean was nearly neutral, the original soil bacterial communities
should be adapted to the neutral environment. While the soil pH of
some terrestrial ecosystems remained nearly neutral, that of some
others gradually acidified/alkalized in the long-term ecosystem
development process (Wu, 1994; Fierer and Jackson, 2006). This
natural acidification/alkalization process was driven by the in-
teractions among climate, organisms and soil, such as by the
decomposition of the plant detritus (Chapin et al., 2011). The
intracellular pH of most microorganisms is generally within one pH
unit of being neutral (Madigan et al., 1997), and the acidification/
alkalization process has resulted in declined soil bacterial diversity.

http://dx.doi.org/10.1016/j.soilbio.2014.11.004
0038-0717/© 2014 Elsevier Ltd. All rights reserved.
276 X. Zhang et al. / Soil Biology & Biochemistry 81 (2015) 275e281
However, the ecological/evolutionary mechanisms of soil pH
affecting bacterial diversity have yet been explored.
In this study, we aim to explore the mechanisms in the process of
long-term soil acidification reducing bacterial diversity across
different land ecosystems. Several processes could contribute to the
relationship between soil bacterial diversity and the natural acidi-
fication pressure. On the one hand, soil acidification will decrease
bacterial diversity through ecological filtering (EF), because the
species not adapted to the acidic pressure will be driven to extinc-
tion. On the other hand, some species may adapt to the acidification
pressure through evolutionary processes. Meanwhile, there are
some species already adapted to the acidification pressure in other
habitats, and they may disperse into the acidified soils and suc-
cessfully establish populations (Martiny et al., 2006; Hanson et al.,
2012). Overall, both evolution and dispersal (ED) may promote the
maintenance of bacterial diversity, counteracting the effect of EF.
Therefore, there were three potential types of mechanism for
different bacterial groups under the soil acidification pressure: I)
neither EF nor ED changes bacterial diversity; II) EF alone reduces
bacterial diversity; III) ED counteracts some effect of EF. Logically, the
bacterial groups driven by the second type of mechanism will be
most vulnerable under the acidification pressure, because they show
no adaptive characteristics. The groups driven by the first and third
types of mechanism will be relatively resistant to changes in pH.
To identify the relative contribution of EF and ED for various soil
bacterial taxonomic groups, we first collected soils with a pH range
of 4e7 from different terrestrial ecosystems across China. This
natural acidification process took place at such a large spatiotem-
poral scale that both EF and ED could be responsible for the pattern
of soil acidification declining bacterial diversity. We also conducted
an acidification experiment with a similar pH range from a neutral
soil. This experimental acidification process took place at a small
spatiotemporal scale such that EF alone could be the primary driver
of the pattern of bacterial diversity, especially for the diversity of
bacterial OTUs (operational taxonomic units) defined as 16S rRNA
gene clusters with larger than 97% sequence similarity
(Stackebrandt and Goebel, 1994). The difference between the nat-
ural and experimental biodiversity patterns should be primarily
caused by ED. Thus, this conceptual framework allows us to
distinguish the three potential types of mechanism by comparing
the patterns observed in the two types of studies. Here we applied
it to identify the mechanism type for the entire bacterial domain as
well as 13 dominant phyla/classes.
2. Materials and methods
2.1. Sampling
To understand the effect of natural soil acidification on bacterial
diversity over a large spatiotemporal scale, we collected soil sam-
ples (with pH 4e7) at 7 sites along the latitude of 43 N and at 17
sites along the longitude of 116 E in the Mainland of China in
August 2009 (Fig. S1; Table S1). At each sampling site, we collected
four soil cores (10 cm depth, 3.5 cm diameter) from four locations
that were at least 3 m apart. The four soil cores were then thor-
oughly mixed.
To examine the effect of soil acidification on bacterial diversity
over a small spatiotemporal scale, we performed a ten-year soil
acidification experiment at the intersection point of the latitudinal
and the longitudinal transects (43 N, 116 E; Fig. S1), with a similar
pH gradient (4e7) as observed in the soil samples collected from
the two transects. The soil at the interaction point was neutral
(~6.90). The experimental design has been described elsewhere
(Zhang et al., 2011). In brief, the study was conducted in a typical
steppe ecosystem near the Inner Mongolia Grassland Ecosystem
Research Station in China, which lies between 43 260 e44 080 N and
116 040 e117 050 E at an average elevation of 1200 m. A continental
middle temperate semiarid climate dominates the area, and is
characterized by a cold, dry winter and a warm, moist summer. The
region is characterized by a dark chest soil. The dominant plant
species accounting for >80% of the total aboveground plant
biomass in the area are Leymus chinensis (Trin.) Tzvel., Stipa grandis
P. Smirn., Agropyron cristatum (L.) Gaertn. and Achnatherum sibir-
icum (L.) Keng. The experimental site (400 m  600 m), constructed
in 1980, was surrounded by an iron fence to exclude animal grazing.
In early July each year from 2000 to 2009, NH4NO3 was added
homogeneously to plots (5 m  5 m) with a 1-m buffer zone at rates
of 0, 1.75, 5.25, 10.5, 17.5, and 28 g N/(m2$yr), respectively. It has
been demonstrated that N addition affected soil bacterial com-
munities primarily through decreasing soil pH in this steppe
ecosystem and that the increase in soil nitrogen content only had a
small effect (Zhang et al., 2011, 2013, 2014; Zhang and Han, 2012).
Each treatment was replicated in three plots. All 18 plots were
distributed across an area of 55 m  110 m in a randomized block
design. In late August 2009, four soil cores (10 cm depth, 3.5 cm
diameter) were collected at four random locations from each plot
and thoroughly mixed.
The pH was measured in 1:2.5 (W/V) suspensions of soil in
distilled water. DNA was extracted from 0.5 g of mixed soil using
the FastDNA SPIN kit for soil (Qbiogene, Carlsbad, CA) according to
the manufacturer's instructions, except that 350 mL of DNA elution
solution was used to elute the DNA in the tenth procedure instead
of 50 mL. The DNA solution was stored at 20  C.
2.2. Measurement of bacterial composition and analysis of
pyrosequence data
The method of 454 pyrosequencing was used to measure the
bacterial composition of each soil sample. The primers 27F (50 -AGA
GTT TGA TCC TGG CTC AG-30 ) and 338R (50 -TGC TGC CTC CCG TAG
GAG T-30 ) were used to amplify the fragment of 16S rRNA gene from
soil DNA. In order to measure bacterial composition of all 42 sam-
ples in one run, a unique 10-mer tag for each soil DNA sample was
added to the 50 -end of the primer 338R (Hamady et al., 2008). Each
20-ml PCR mixture contained 4 ml FastPfu Buffer (5; Transgen), 2 ml
of 2.5 mM dNTPs, 0.4 ml of each primer (5 mM), 0.8 ml of DNA tem-
plate, and 0.4 ml of FastPfu Polymerase (Transgen). The PCR protocol
was 95  C for 2 min (denature); 25 cycles of 95  C for 30 s (dena-
ture), 55  C for 30 s (anneal), 72  C for 30 s (elongate); and 72  C for
5 min. Three PCRs were performed for each sample. The combined
products were purified by agarose gel electrophoresis and recov-
ered. The recovered products were quantified with PicoGreen using
a TBS-380 Mini-Fluorometer, and equal molar concentrations of
PCR products for each sample were pooled. The pooled products
were sequenced in a Roche 454 Genome Sequencer FLX Titanium
system at Shanghai Majorbio Bio-pharm Technology Co., Ltd.
The pyrosequence reads were analyzed using the Mothur soft-
ware package (Version 1.19) (Schloss et al., 2009). The reads were
first assigned to samples according to their tags, and those <150 bp
in length or with ambiguous characters were removed. The
chimeric sequences were excluded by the chimera.uchime com-
mand with default parameters. The first 150 bp of the remaining
reads were aligned to the Silva database (Version 106) (Pruesse
et al., 2007), and non-bacterial reads were further removed. To
minimize the influence of unequal sampling on the subsequently
calculated indexes, 3007 reads were randomly selected from each
sample for analysis. All the 126,294 (3007  42) sequences were
clustered into OTUs based on 97% similarity (Stackebrandt and
Goebel, 1994). The OTU number of each sample was used to
represent the richness of the entire bacterial domain. A different
 X. Zhang et al. / Soil Biology & Biochemistry 81 (2015) 275e281
approach was adopted to calculate the OTU richness of the 13
dominant bacterial phyla/classes. For each phylum/class, we
randomly selected 30 representative sequences from a sample,
calculated the OTU number represented by the 30 sequences,
repeated this process 1000 times, and used the average OTU
number to represent the richness. For each of the 14 bacterial
taxonomic groups (including the entire bacterial domain) in both
natural and experimental soils, we further tested whether there
were linear relationships between OTU richness and soil pH with
SPSS software (SPSS 13.0 for WINDOWS).
For each of the 14 bacterial groups, we calculated the
BrayeCurtis distance based on the abundance of OTUs to represent
the compositional variation among localities (Bray and Curtis,
1957). Mantel test was used to analyze whether the latitudinal
and longitudinal differences affected the bacterial compositional
variation (Bonnet and Peer, 2002). None of the 14 taxonomic groups
showed significant positive response (r > 0 and P < 0.05; Table S2),
suggesting that none of them was dispersal-limited. In other words,
bacterial species already adapted to other acidic habitats might
have colonized these acidified soils through random dispersal.
2.3. Conceptual framework
To differentiate the three types of mechanism (see the third
paragraph of the introduction), we proposed a conceptual
framework. During the long-term development of various eco-
systems along the two sampling transects, soils that were origi-
nally neutral were found to have been gradually acidified from
west to east and from north to south (Fig. S1; Table S1) (Wu,
1994). This process has taken place slowly over a long time span
of perhaps tens of millions of years (Chapin et al., 2011), implying
that bacterial diversity might have been driven by both EF and ED.
For the ten-year soil acidification experiment, however, the time
scale was negligibly small; although genetic changes might take
place in some rapidly evolving genes, the 16S rRNA gene was so
conservative that evolution could not have had a noticeable effect
on the diversity of OTUs based on 16S rRNA gene sequences
(Stackebrandt and Goebel, 1994). Meanwhile, the soil acidification
experiment was conducted in a large steppe ecosystem with only
neutral soils, and the effect of species dispersal from acidic en-
vironments in other ecosystems would be presumably small.
Since the effect of ED is therefore negligible, changes in bacterial
diversity due to soil acidification could be mainly due to EF.
Meanwhile, it seems reasonable to assume that, if EF occurred in
the experimental soils, it would surely have occurred over the
longer time intervals in the natural soils. If EF did not occur in the
natural soils, it would not occur over the shorter time intervals in
the experimental soils.
Table 1
The three types of mechanism of EF (ecological filtering) and ED (evolution/dispersal) co-driving bacterial diversity in the experimental and natural soils.
The type of mechanism Cases Experimental
samples
EF
I 1 e e
II 2 e
II 3 Y
II 4 Y
III 5 e
III 6 Y
III 7 Y
I, II and III represent the three types of mechanism (see the details in the third paragraph of the Introduction). “e” means that EF or ED has no effect. “Y” means that EF
decreases OTU richness. “[” means that ED promotes OTU richness. The size of “Y” and “[” represent the intensity of these effects.
277
The three types of mechanism will generate different patterns of
bacterial OTU diversity vs. soil pH (Table 1; Fig. 1). The first type of
mechanism means that neither EF nor ED operates in the natural
soils, and thus they will not operate in the experimental soils,
either. In this case (case 1), as soil pH decreases, bacterial OTU
richness does not change in either the experimental or the natural
soils (Fig. 1a1). The second type of mechanism means that only EF
operates in the natural soils, and there are three possible cases
(cases 2, 3 and 4; Table 1). In case 2, EF operates in the natural soils
but not in the experimental soils, leading to the pattern that soil
acidification decreases OTU richness in the natural soils but not in
the experimental soils (Fig. 1b1). In case 3, EF operates in the nat-
ural soils as well as in the experimental soils, resulting in soil
acidification declining OTU richness equally in both soils (Fig. 1b2).
In case 4, EF has a larger effect on OTU richness in the natural soils
than in the experimental soils, so the OTU richness decreases to a
larger extent in the natural soils than in the experimental soils
(Fig. 1b3). The third type of mechanism means that ED counteracts
some effect of EF in the natural soils, and there are three possible
cases (cases 5, 6 and 7; Table 1). In case 5, while ED counteracts all
the effect of EF in the natural soils, EF does not operate in the
experimental soils, leading to the pattern that OTU richness does
not change in both soils (Fig. 1c1). In case 6, while ED counteracts
some effect of EF in the natural soils, EF operates in the experi-
mental soils, generating the pattern that soil acidification reduces
OTU richness more slowly in the natural soils than in the experi-
mental soils (Fig. 1c2). In case 7, ED counteracts all effect of EF in the
natural soils and EF operates in the experimental soils. As soil pH
decreases, OTU richness remains constant in the natural soils but
declines in the experimental soils (Fig. 1c3).
While cases 1 and 5 produce indistinguishable pattern of OTU
richness vs. soil pH (Fig. 1a1 and c1), all the other five cases can be
distinguished according to their patterns (Fig. 1b1, b2, b3, c2 and
c3), enabling us to infer the relative contribution of ED and EF in
driving OTU richness under the long-term natural acidification
pressures. slopeE and slopeN were used to represent the slope of the
linear relationship between OTU richness and soil pH in the
experimental and natural soils, respectively. In Fig. 1b1, slopeE
should be non-significant, but slopeN should be significant
(P < 0.05). Both slopeE and slopeN should be significant in Fig. 1b2,
b3 and c2, and slopeN should be equal to, larger than and smaller
than slopeE, respectively. In Fig. 1c3, slopeE should be significant,
but slopeN should be non-significant.
We could further develop a quantitative index to calculate the
relative contribution of EF and ED in the natural soils for the five
cases. While slopeE represents the intensity of EF in the experi-
mental soils, slopeN represents the balance between EF and ED in
the natural soils. Because EF and ED have opposite effects on OTU
E N
slope slope
Natural samples Corresponding E
slope
pattern in Fig. 1
ED EF ED
e e a1
e Y e b1 <0
e Y e b2 ¼0
e e b3 <0
e Y [ c1
e Y c2 0e1
e Y [ c3 ¼1
278 X. Zhang et al. / Soil Biology & Biochemistry 81 (2015) 275e281
Fig. 1. The theoretical patterns of bacterial OTU richness vs. soil pH in experimental (black) and natural (red) soils. a1, b1eb3 and c1ec3 correspond to the mechanism type of I, II
and III, respectively (see Table 1). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
richness, we can use (slopeE  slopeN) to represent the intensity of
ED. Then, ((slopeE  slopeN)/slopeE) can indicate the relative
contribution of ED and EF. There was a theoretical interval for each
of the five cases (Table 1), e.g., it should be between 0 and 1 in case
6 when ED counteracts only part effect of EF. Overall, the interval
should be 0 and 0e1 for the II and III type of mechanism,
respectively (Table 1). In practice, the 95% confidence interval of
this index could be estimated with Monte-Carlo simulation.
We used this framework to investigate the relative contribution
of EF and ED in driving the OTU richness of 14 bacterial taxonomic
groups, namely the entire bacterial domain, six dominant phyla,
and seven dominant classes. We also explained the influence of
other ecological factors (e.g. soil heterogeneity, nutrient content
and vegetation type) on the effectiveness of this framework in the
discussion.
2.4. Accession numbers
The sequence reads for all samples have been deposited in the
National Center for Biotechnology Information Sequence Reads
Archive (accession no. SRA057671).
3. Results
Clustering all 126,294 sequences into OTUs with 97% similarity
yielded an average of 1258 OTUs for each sample and a total of
26,978 OTUs for all 42 samples.
The OTU richness of the entire bacterial domain showed sig-
nificant linear relationship with soil pH in both the experimental
and natural soils, and slopeN (108) was smaller than slopeE (188;
Fig. 2a). This pattern was similar to the theoretical pattern in
Fig. 1c2. The value of ((slopeE  slopeN)/slopeE) of the entire bac-
terial domain was 0.42, with the 95% confidence interval of
0.33e0.50 (Table 2). This result suggests that the third type of
mechanism was operating (Table 1).
The patterns of Acidobacteria phylum and Deltaproteobacteria
class (Fig. 2b, n) were also similar to the theoretical pattern in
Fig. 1c2. The OTU richness of four phyla (Actinobacteria, Bacter-
oidetes, Chloroflexi and Planctomycetes) and three classes (Acid-
imicrobidae, Actinobacteridae and Gammaproteobacteria) showed
significant linear relationships with soil pH in the experimental
soils but non-significant relationships in the natural soils (Fig. 2cef,
h, i, m), which was similar to the theoretical pattern in Fig. 1c3. The
values of ((slopeE  slopeN)/slopeE) for all the nine phyla/classes
were between 0 and 1 (Table 2), indicating that they were governed
by the third mechanism type.
The OTU richness of three bacterial taxonomic groups (the
entire Proteobacteria phylum, and the Alpha- and Beta-
proteobacteria classes) showed non-significant linear relationship
with soil pH in the experimental soils but significant relationship in
the natural soils (Fig. 2g, k, l). This pattern was similar to the
theoretical pattern in Fig. 1b1. Meanwhile, the values of
((slopeE  slopeN)/slopeE) for all of them were negative (Table 2),
showing that the second type of mechanism was operating.
The OTU richness of the Rubrobacteridae class showed non-
significant linear relationship with soil pH in both the experi-
mental and natural soils (Fig. 2j). This pattern was similar to the
theoretical pattern in Fig. 1a1 and c1, so the first or the third type of
mechanism might be operating (Table 2).
4. Discussion
Overall, our results demonstrated that different bacterial taxo-
nomic groups were driven by different types of mechanism.
Meanwhile, the third type of mechanism, that ED counteracted at
least some effect of EF, was the dominant mechanism. This type of
mechanism influenced the OTU richness of 10 out of the 14 bac-
terial taxonomic groups (Table 2).
The entire bacterial domain was driven by the third type of
mechanism. In particular, the value of ((slopeE  slopeN)/slopeE)
 X. Zhang et al. / Soil Biology & Biochemistry 81 (2015) 275e281 279

Fig. 2. The actual patterns of OTU richness vs. soil pH in experimental (black) and natural (red) soils for 14 bacterial taxonomic groups. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
280 X. Zhang et al. / Soil Biology & Biochemistry 81 (2015) 275e281

Table 2
The relative contribution of EF and ED in driving the OTU richness of the 14 bacterial taxonomic groups.

Bacterial group Relative Actual pattern SlopeE SlopeN E

slope slope
N
Theoretical Corresponding
E
abundance (%)a in Fig. 2 slope pattern in Fig. 1 mechanism type
(95% confidence interval)

Domain Bacteria 100 a 188b 108b 0.42 (0.33e0.50) c2 III

Phylum Acidobacteria 10.2 b 3.02b 0.79b 0.74 (0.69e0.78) c2 III

Actinobacteria 38.4 c 0.63b 0.34 0.46 (0.35e0.55) c3 III
Bacteroidetes 3.1 d 6.29b 0.81 0.87 (0.85e0.89) c3 III
Chloroflexi 7.0 e 1.12b 0.47 0.58 (0.50e0.65) c3 III
Planctomycetes 3.3 f 1.66b 0.0068 0.996 (0.995e0.997) c3 III
Proteobacteria 23.0 g 0.95 1.66b 0.75 (1.06~0.46) b1 II

Class Acidimicrobidae 2.1 h 2.44b 0.89 0.64 (0.57e0.69) c3 III

Actinobacteridae 21.3 i 0.54b 0.23 0.57 (0.49e0.65) c3 III
Rubrobacteridae 12.8 j a1 or c1 I or III
Alphaproteobacteria 13.8 k 0.38 1.48b 2.88 (3.55~2.19) b1 II
Betaproteobacteria 3.3 l 0.86 1.39b 0.62 (0.91~0.36) b1 II
Gammaproteobacteria 2.5 m 3.73b 0.10 0.973 (0.968e0.978) c3 III
Deltaproteobacteria 2.9 n 2.08b 0.79b 0.62 (0.56e0.68) c2 III
a
Represents the mean relative abundance in all the experimental and natural samples. slopeE and slopeN represent the slopes of experimental and natural soils, respectively
(see the details in Fig. 2).
b
Means that the linear relationship was statistically significant (P < 0.05).

was 0.42 (Table 2), suggesting that nearly a half of the effect of EF
was counteracted by ED. However, this result did not mean that the
relative contribution of EF and ED was the same for all the phyla
within the bacterial domain. In fact, the Proteobacteria phylum was
driven by EF alone (the second mechanism type). Although the
phyla of Bacteroidetes and Planctomycetes were also driven by the
third mechanism type, they had large ((slopeE  slopeN)/slopeE)
values (0.87 and 0.996, respectively; Table 2), meaning that almost
all the effect of EF was counteracted by ED. Similarly, while the
entire Proteobacteria phylum was governed by the second type of
mechanism, Gamma- and Delta-proteobacteria was driven by the
third type of mechanism (Table 2).
These bacterial taxonomic groups were driven by different types
of mechanism, suggesting that they had different fitness under the
natural acidification pressure. Unfortunately, soil acidification was
further intensified by current acidic rain and N deposition (Clack
and Tilman, 2008; Zhang et al., 2011). The groups being driven by
the third type of mechanism were adaptive evolutionarily or they
had a high dispersal, so they would be resistant to the acidic se-
lective pressure. In contrast, the groups being driven by the second
type of mechanism (e.g. the Alpha- and Beta-proteobacteria) were
not adaptive and would be more vulnerable to acidification (Lauber
et al., 2009; Rousk et al., 2010). Betaproteobacteria play an impor-
tant role in oxidizing ammonium (Horz et al., 2004; Martiny et al.,
2011), and our result implies that this ecosystem function is
vulnerable in the acidic soils. In contrast, when the acidified soils
were neutralized by added lime, the groups being driven by the
third type of mechanism would be vulnerable and the groups by
the second type would be favored.
We have identified the relative contribution of EF and ED in
driving soil microbial diversity using a simple conceptual frame-
work, which we hope would stimulate the investigation of their
relative contribution to processes of other ecological factors (e.g.
precipitation and temperature) driving soil microbial diversity.
However, it is important to note that our conceptual framework
focuses on acidification as the main driver of soil bacterial diversity,
and that other factors might affect the effectiveness of the frame-
work. Firstly, the current neutral soil community was used to
represent the primitive neutral soil community originated from the
ocean, but the long-term ecological and evolutionary processes
might have caused the former to be more diverse than the latter.
This diversification process was primarily due to the high hetero-
geneity of soil environment compared to the ocean environment
(Rainey and Travisano, 1998; Horner-Devine et al., 2004; Ranjard
et al., 2013), and this process would happen in soils with all types of
pH, regardless of being neutral or acidic. Soil heterogeneity would
increase the value of the y-intercept in the linear relationship of
bacterial diversity vs. soil pH. However, in this research, we aimed
to quantify the effect of ED induced by soil acidification rather than
by soil heterogeneity, and the effect caused by soil acidification was
exhibited in the slope (rather than the y-intercept) of the linear
relationship of bacterial diversity vs. soil pH (Fig. 1). Therefore, the
increased environmental heterogeneity would not have significant
influence on our results.
Secondly, although soil pH alone accounted for a large propor-
tion of the variation in soil bacterial diversity across different
terrestrial ecosystems and it integrated the effect of many ecolog-
ical factors (e.g. nutrient availability, vegetation type and soil
moisture (Fierer and Jackson, 2006)), other factors (e.g. trace
element content) might still have some effect on soil bacterial di-
versity. If this is indeed found to be the case, the linear relationship
between bacterial diversity and soil pH is likely to be weakened,
with soil pH playing a lesser role (in terms of R2) in the linear
relationship. However, the contribution of EF and ED was quantified
from the slope of bacterial diversity vs. soil pH rather than the R2
value. Thus, these factors would have limited influence on our
results.
Finally, the relationship between OTU diversity and soil pH in
the experimental soils was assumed to be mainly driven by EF in
our conceptual framework. However, some evolution and dispersal
might still have occurred over the ten-year time scale. Meanwhile,
there might be rare acid-tolerant bacterial species present in the
neutral experimental soils, which would become common under
acidification. All these factors would partly counteract the effect of
EF in the experimental soils. In other words, when slopeE was used
to represent the effect of EF in our conceptual framework, the actual
effect of EF and thus the ((slopeE  slopeN)/slopeE) value may have
been underestimated. However, given the difficulty of quantifying
in-situ evolution and dispersal, our approach provides a first
approximation of how EF and ED combine to regulate soil bacterial
communities.
Acknowledgments
We thank Dengyun Wu, Yaxing Xie and Yalin Xie for help in
sampling; Yongfei Bai, Lixia Zhang and many others for help in
 X. Zhang et al. / Soil Biology & Biochemistry 81 (2015) 275e281
conducting the soil acidification experiment; Qiuping Hu, Guo Yu
and many others in Shanghai Majorbio Bio-pharm Technology Co.,
Ltd. for help in pyrosequencing; Professor Frederick Cohan of
Wesleyan University, Professor Kostas T. Konstantinidis and Dr. Jiaqi
Tan of Georgia Institute of Technology for making comments on the
early drafts. This work was supported by the National Natural Sci-
ence Foundation of China (31300431), the “Strategic Priority
Research Program” of the Chinese Academy of Sciences
(XDB15010404), State Key Laboratory of Forest and Soil Ecology of
China (Grant No. LFSE2013-15), and National Science Foundation of
USA (Grant No. DEB-1257858 and DEB-1342754).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.soilbio.2014.11.
004.
References
Bonnet, E., Peer, Y.V., 2002. zt: a software tool for simple and partial Mantel tests.
Journal of Statistical Software 7, 1e12.
Bray, J.R., Curtis, J.T., 1957. An ordination of the upland forest communities of
Southern Wisconsin. Ecological Monograph 27, 325e349.
Chapin III, F.S., Matson, P.A., Vitousek, P.M., 2011. Principles of Terrestrial Ecosystem
Ecology, second ed. Springer, New York.
Clack, C.M., Tilman, D., 2008. Loss of plant species after chronic low-level nitrogen
deposition to prairie grassland. Nature 451, 712e715.
Fierer, N., Jackson, R.B., 2006. The diversity and biogeography of soil bacterial
communities. Proceedings of the National Academy of Sciences of the United
States of America 103, 626e631.
Hamady, M., Walker, J.J., Harris, J.K., Gold, N.J., Knight, R., 2008. Error-correcting
barcoded primers for pyrosequencing hundreds of samples in multiplex. Nature
Methods 5, 235e237.
Hanson, C.A., Fuhrman, J.A., Horner-Devine, M.C., Martiny, J.B., 2012. Beyond
biogeographic patterns: processes shaping the microbial landscape. Nature
Reviews Microbiology 10, 497e506.
Horner-Devine, M.C., Carney, K.M., Bohannan, B.J.M., 2004. An ecological perspec-
tive on bacterial biodiversity. Proceedings of the Royal Society B: Biological
Sciences 271, 113e122.
Horz, H.P., Barbrook, A., Field, C.B., Bohannan, B.J.M., 2004. Ammonia-oxidizing
bacteria respond to multifactorial global change. Proceedings of the National
Academy of Sciences of the United States of America 101, 15136e15141.
Jones, R.T., Robeson, M.S., Lauber, C.L., Hamady, M., Knight, R., Fierer, N., 2009.
A comprehensive survey of soil acidobacterial diversity using pyrosequencing
and clone library analyses. The ISME Journal 17, 1e12.
281
Lauber, C.L., Hamady, M., Knight, R., Fierer, N., 2009. Pyrosequencing-based
assessment soil pH as a predictor of soil bacterial community structure at the
continental scale. Applied and Environmental Microbiology 75, 5111e5120.
Madigan, M., Martinko, J., Parker, J., 1997. Brock Biology of Microorganisms. Prentice
Hall, Upper Saddle River, NJ.
Martin, W., Baross, J., Kelley, D., Russell, M.J., 2008. Hydrothermal vents and the
origin of life. Nature Reviews Microbiology 6, 805e814.
Martiny, J.B., Bohannan, B.J., Brown, J.H., Colwell, R.K., Fuhrman, et al., 2006. Mi-
crobial biogeography: putting microorganisms on the map. Nature Reviews
Microbiology 4, 102e112.
Martiny, J.B., Eisen, J.A., Penn, K., Allision, S.D., Horner-Devine, M.C., 2011. Drivers of
bacterial beta-diversity depend on spatial scale. Proceedings of the National
Academy of Sciences of the United States of America 108, 7850e7854.
Nisbet, E.G., Sleep, N.H., 2001. The habitat and nature of early life. Nature 409,
1083e1091.
Prosser, J.I., Bohannan, B.J.M., Curtis, T.P., Ellis, R.J., Firestone, M.K., Freckleton, R.P.,
Green, J.L., Green, L.E., Killham, K., Lennon, J.J., Osborn, A.M., Solan, M., van der
Gast, C.J., Young, J.P.W., 2007. The role of ecological theory in microbial ecology.
Nature 5, 384e392.
Pruesse, E., Quast, C., Knittel, K., Fuchs, B.M., Ludwig, W., et al., 2007. SILVA: a
comprehensive online resource for quality checked and aligned ribosomal RNA
sequence data compatible with ARB. Nucleic Acids Research 35, 7188e7196.
Rainey, P.B., Travisano, M., 1998. Adaptive radiation in a heterogeneous environ-
ment. Nature 394, 69e72.
Ranjard, L., Dequiedt, S., Pre vost-Boure , N.C., Thioulouse, J., Saby, N.P.A., et al., 2013.
Turnover of soil bacterial diversity driven by wide-scale environmental het-
erogeneity. Nature Communications 4, 1434.
Rousk, J., Bååth, E., Brookes, P.C., Lauber, C.L., Lozupone, C., et al., 2010. Soil bacterial
and fungal communities across a pH gradient in an arable soil. The ISME Journal
4, 1340e1351.
Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., et al., 2009. Intro-
ducing mothur: open-source, platform-independent, community-supported
software for describing and comparing microbial communities. Applied and
Environmental Microbiology 75, 7537e7541.
Stackebrandt, E., Goebel, B.M., 1994. Taxonomic note: a place for DNA-DNA reas-
sociation and 16S rRNA sequence analysis in the present species definition in
bacteriology. International Journal of Systematic Bacteriology 44, 846e849.
Wu, D.S., 1994. The Atlas of Soil Environmental Background Value in the People's
Republic of China. China Environmental Science Press, Beijing.
Zhang, X.M., Chen, Q.S., Han, X.G., 2013. Soil bacterial communities respond to
mowing and nutrient addition in a steppe ecosystem. PLoS ONE 9, e89711.
Zhang, X.M., Han, X.G., 2012. Nitrogen deposition alters soil chemical properties and
bacterial communities in the Inner Mongolia grassland. Journal of Environ-
mental Sciences 24, 1483e1491.
Zhang, X.M., Liu, W., Bai, Y.F., Zhang, G.M., Han, X.G., 2011. Nitrogen deposition
mediates the effects and importance of chance in changing biodiversity. Mo-
lecular Ecology 20, 429e438.
Zhang, X.M., Wei, H.W., Chen, Q.S., Han, X.G., 2014. The counteractive effects of
nitrogen addition and watering on soil bacterial communities in a steppe
ecosystem. Soil Biology and Biochemistry 72, 26e34.